This proposal seeks funds to examine the fundamental cellular behavior of nitric oxide (NO) and S- nitrosothiols (RSNO). Nitric oxide (NO) is an essential component of vascular function in pulmonary and all other tissues. In the lung, NO has particular importance as inhaled NO is used as a therapy for pulmonary hypertension, and alterations in NO homeostasis have been appreciated in many pulmonary diseases including asthma and cystic fibrosis. In addition S-nitrosoglutathione (GSNO) has been detected in micro molar concentration in lung lavage and is decreased in asthma. In general RSNO have been invoked as 'carriers'of NO bioactivity and RSNO have been used in cell culture studies as 'NO-donors'. Our studies in the previous funding period have highlighted the fact that NO and RSNO affect cellular functions by clearly different mechanisms. In particular we characterized the mechanisms by which RSNO can be transported into cells and have shown that these compounds can affect cellular functions via both NO-dependent and NO-independent pathways. This has led to the overall hypothesis of this proposal, that lmwtRSNO formation represents a bifurcation in NO signaling. We will address this hypothesis by the following three Specific Aims. 1) We will further characterize how lmwtRSNO are synthesized and transported into cells and how the glutathione/NADH/NADPH can control the activity and stability of RSNO. 2) Examine the differential sensitivity of selected pathways to modulation by RSNO and NO. 3) Define modifications of intracellular proteins using a range of lmwtRSNO and NO concentrations to uncover the differential sensitivity of proteins and pathways to S-nitrosation and S-thiolation. We will accomplish these aims using lung-cell specific cell culture models, specialized chemiluminescence detection methodologies and proteomic techniques. Successful completion of these studies will disentangle the cellular effects of NO from the direct effects of RSNO to more fully understand how these species affect cellular function. Importantly, these studies will point to a role of RSNO formation not simply as 'carriers'of NO bioactivity but as a distinct bifurcation in NO signaling pathways.